Hox homeodomain transcription factors play essential roles in vertebrate limb patterning and morphogenesis, but the mechanisms of Hox regulation remain elusive at the molecular level. The Pbx1 TALE homeodomain protein is a homolog of Drosophila extradenticle (Exd), which has critical roles in patterning of the fly body. While the fly has only one TALE-encoding gene, Exd, the mouse has four Pbx genes (Pbx1-4). Based on molecular and biochemical analyses, for the last fifteen years the prevailing view has been that Pbx/Exd primarily helps Hox proteins execute their developmental programs. Hence, Pbx proteins have been named as "Hox cofactors". However, it has been unclear whether Pbx proteins exert their roles more broadly than Hox ancillary factors in vivo. Our objectives are to: 1) use the limb as a tractable system to delineate genetic and molecular networks controlled by TALE proteins in developmental programs;2) establish whether regulation of Hox "collinear" expression in the limb, an elegant but mysterious developmental phenomenon, is mediated by Pbx TALE homeodomain proteins. Using the mouse as a system, we have established that different Pbx genes share partially overlapping roles in various developmental processes, including limb patterning. Accordingly, Pbx1/Pbx2 double homozygous (Pbx1-/-;Pbx2-/-) embryos lack limbs altogether, while Pbx1-/-;Pbx2 mutants exhibit dramatic limb truncations. Most significantly, we have uncovered that during limb morphogenesis Pbx1/Pbx2 control the onset and spatial distribution of 5'Hox expression in the autopod. Thus, Pbx proteins hierarchically govern Hox gene expression in this system. In view of these unanticipated findings, our current working hypothesis proposes that Pbx homeoproteins do not function exclusively as Hox ancillary factors in the limb, but that they control Hox genes and possibly regulate 5'HoxD expression at the transcriptional level in the limb bud. We will test our hypothesis in the mouse using genetic and molecular approaches. First, by tissue-specific and inducible genetic ablation, using our new Pbx1 conditional allele (on a Pbx2-deficient background), and available Cre lines (one of which is inducible in the mesenchyme), we will dissect Pbx1/Pbx2 spatial and temporal requirements in limb field and bud mesenchyme. We will then determine when Hox expression is first affected by Pbx1/2 loss in the mesenchyme. By molecular approaches, we will subsequently determine whether Pbx1/2 regulate 5'HoxD transcription by direct control of the HoxD GCR, a genomic region that controls HoxD collinear expression in the autopod. Furthermore, we will test whether Pbx binding to the HoxD GCR has functional bearings on transcription by both transient transfections in cell culture and transient transgenesis experiments in vivo in the mouse. Finally, we will analyze Pbx1/Pbx3 phenotypic interactions in mouse forelimb (FL) development and assess Pbx1/Pbx3 control of early Hox expression in the FL field. We will specifically focus on FL patterning since Pbx3, which is not expressed in hindlimbs, acts as a FL-specific marker. Completion of these studies will shed light on Pbx- controlled programs in limb development and establish novel regulatory networks that govern transcription of Hox genes, key architects of the body plan. Also, this work will bear directly on our understanding of human genetic limb malformations. More broadly, given the involvement of HOX genes in human chromosomal translocations that perturb HOXD13 or HOXA9 expression resulting in acute leukemia, our studies will inform general comprehension of Hox gene function also in other contexts, as human neoplastic transformation. PUBLIC HEALTH RELEVANCE: Homeodomain proteins control patterning and morphogenesis of the appendicular skeleton within genetic and molecular networks that are poorly understood. We have established critical genetic roles for the TALE-class of homeoproteins Pbx1 and Pbx2 in limb development. The studies proposed in this application will provide novel insight into the control of HoxD gene expression by Pbx in the mesenchyme of the distal limb, as well as strengthen our new model wherein Pbx do not act solely as Hox ancillary factors in limb development, but govern Hox gene expression. Furthermore, the proposed experiments will shed light on the unique and partially overlapping contributions of Pbx1/Pbx2 in limb bud mesenchyme in skeletal development. Finally, the planned studies will establish as yet unknown roles for Pbx3, a forelimb-specific marker, in patterning anterior and proximal forelimb bud mesenchyme together with Pbx1, as well as uncover novel molecular networks that are perturbed when Pbx1/Pbx3 are concomitantly lost. Direct involvement of Hox genes in human congenital malformations has been reported as a result of genetic mutations. Accordingly, a broader impact of this work will be the generation of new knowledge on the pathogenesis of human congenital disorders, including those that affect limb skeletal development and function. Indeed, mutations in 5'HOX genes result in severe limb abnormalities, including synpolydactyly, monodactyly, club foot malformation, hand-foot-genital syndrome, and brachydactyly, among others. Additionally, common forms of human acute myelogenous leukemias with fatal outcome involve chromosomal translocations that join HOXD13 or HOXA9 to NUP98, thereby resulting in perturbed and ectopic expression of the HOX gene. Lastly, perturbed expression of HOX genes in humans has been reported in several types of cancers, including lung cancers, gliomas, neuroblastomas, and breast and ovarian cancers. While this proposal focuses on Pbx functions in the limb and potential regulatory roles of Pbx on Hox transcription in this system, perturbation of Pbx-ruled developmental processes in the mouse leads to a variety of other abnormalities (in addition to craniofacial, axial, rib cage, limb, and girdle defects) that closely model a broad range of human congenital diseases, including congenital heart defects, diabetes mellitus, congenital asplenia. Therefore, it is of high social and medical relevance to conduct basic research that will establish as yet unknown Pbx-controlled networks in organogenesis as well as novel mechanisms for Hox transcriptional regulation and collinear expression. Deeper comprehension of these basic processes in the limb will be applicable to other contexts within the developing embryo and ultimately aid our understanding of the pathogenesis of human congenital diseases as well as neoplastic transformation.